This application claims priority from European Patent Application No. 00126019.9, filed in English on Nov. 28, 2000, the disclosure of which is incorporated by reference herein in its entirety.
Indocyanine dyes conform to the generalised formula: 
where R11, R12, R13, R14, R21, R22, R23 and R24 are either hydrogens or substituents; the substituent couples R13, R14 and/or R23, R24 can form a condensed benzene ring, in turn bearing substituents; n=1-3; Ln1, Ln2 are either methines (Cxe2x80x94H) or substituted methines (Cxe2x80x94R).
Comprehensive reviews regarding indocyanine dyes have been written by Frances M. Hamer, xe2x80x9cThe Chemistry of Heterocyclic Compoundsxe2x80x9d, vol. 18, xe2x80x9cThe Cyanine Dyes and Related Compoundsxe2x80x9d, Weissberger Ed., Wiley Interscience, New York, (1964); D. M. Sturmer, xe2x80x9cThe Chemistry of Heterocyclic Compoundsxe2x80x9d, xe2x80x9cSpecial Topics in Heterocyclic Chemistryxe2x80x9d, chapter VIII, xe2x80x9cSynthesis and Properties of Cyanine and Related Dyesxe2x80x9d, Weissberger Ed., Wiley, N.Y., (1977); xe2x80x9cThe Kirk-Othmer Encyclopaedia of Chemical Technologyxe2x80x9d vol. 7, p. 782, xe2x80x9cCyanine Dyesxe2x80x9d, Wiley, N.Y., (1993).
For many years, indocyanine dyes have been very useful as sensitisers in photography, especially in the red and near infrared regions of the spectrum. However, in more recent years, there has been an upsurge of new uses of these dyes in innovative technological areas, such as laser and electro-optic applications, optical recording media, medical, biological and diagnostic. These new applications of indocyanine dyes place high demands on the degree of purity required, and the reproducibility of synthetic methods and purification steps is very important. These requirements are especially stringent for dyes designed to improve detection of ribonucleic acid (RNA), deoxyribonucleic acid (DNA) and of antigens in immunoassays. In these fields, the trend toward an increasing miniaturisation is accompanied by an increasing demand on sensitivity of the reporter molecules or labels. One way to increase the sensitivity of conventional fluorescence method is to use laser sources for the excitation. However, traditional fluorescent labels based on fluoresceins or rhodamines required expensive and/or bulky lasers. Moreover, their fluorescence occurs in the blue-green to green regions of the visible spectrum, where interference from the sample matrix is more likely to occur. Indocyanine dyes do not suffer from these limitations. They can be efficiently excited by means of small, inexpensive solid state devices such as laser diodes or light emitting diodes, with extinction coefficients often several times higher than fluoresceins and rhodamines; they emit in the red and near-infrared regions of the spectrum, where non-specific fluorescence from the sample is low or lacking; another sources, Raman noise, becomes smaller with the inverse fourth power of wavelength.
To be useful as a label, a dye has to be provided with a suitable side chain containing a functional group. While the main part of the dye structure is generally known from previous applications, the introduction of a functional group into the structure for the purpose of conjugation, or binding to another molecule, represents the innovative step in the inventions concerning the use of the dye as a labelling reagent. In general, only one such functionalised side arm is preferable, in order to avoid cross-linking or purification problems. With a few exceptions, limited to heptamethine dyes, the standard approach in the design of indocyanine labelling reagents has been to attach the functionalised side arm to one of the heterocyclic nuclei of the dye:
HET1xe2x80x94HET2xe2x80x94Z 
See, for instance: J. S. Lindsey, P. A. Brown, and D. A. Siesel, xe2x80x9cVisible Light-Harvesting in Covalently-Linked Porphyrin-Cyanine Dyesxe2x80x9d, Tetrahedron, 45, 4845, (1989); R. B. Mujumdar, L. A. Ernst, Swati R. Mujumdar, C. J. Lewis, and A. S. Waggoner, xe2x80x9cCyanine Dye Labelling Reagents: Sulfoindocyanine Succinimidyl Estersxe2x80x9d, Bioconjugate Chemistry, 4, 105, (1993); G. Mank, H. T. C. van der Laan, H. Lingeman, Cees Goojer, U. A. Th. Brinkman, and N. H. Velthorst, xe2x80x9cVisible Diode Laser-Induced Fluorescence Detection in Liquid Chromatography after Precolumn Derivatization of Aminesxe2x80x9d, Anal. Chem., 67, 1742, (1995).
The general synthetic strategy necessary to prepare these labelling reagents is as follows. First, a quaternised nitrogen heterocycle HET1 is prepared. Then, this heterocyclic base is reacted with an electrophilic reagent such as PhNHxe2x80x94(CHxe2x95x90CH)nxe2x80x94CHxe2x95x90NHPh.HCl or ROxe2x80x94(CHxe2x95x90CH)nxe2x80x94CH(OR)2, where Ph is a phenyl ring and R a methyl or ethyl group, to obtain a so-called hemicyanine dye, HET1xe2x80x94(CHxe2x95x90CH)nNHPh/HET1xe2x80x94(CHxe2x95x90CH)nNAcPh, where Ac is the acetyl radical or HET1xe2x80x94(CHxe2x95x90CH)nxe2x80x94OR. These intermediates are then reacted with a different quaternary nitrogen heterocycle, HET2. The functionalised side arm can be attached either to the first or to the second quaternised nitrogen heterocycle. The final result is an asymmetric polymethine labelling reagent, HET1xe2x80x94(CHxe2x95x90CH)nxe2x80x94HET2xe2x80x94Z.
Unfortunately, the hemicyanine intermediates are notoriously difficult to obtain in good yields and/or in a pure form. For example, the condensation of N-methyl-2,3,3-trimethyl[3H]indolium iodide with malonaldehyde dianil monochloride in acetic anhydride is said (Piggott and Rodd, BP 355,693/1930) to give rise to a green intermediate, indicating a strong contamination of the desired, yellow hemicyanine intermediate (yellow) with symmetric, blue indocyanine dye, FIG. 2. Moreover, when F. M. Hamer, in xe2x80x9cSome Unsymmetrical Pentamethincyanine Dyes and their Tetramethin Intermediatesxe2x80x9d tried to prepare a pure sample of the same hemicyanine intermediate, obtained it in an 8% yield, after a lengthy and wasteful procedure based on multiple extractions and precipitations. More recently, R. B. Mujumdar, L. A. Ernst, Swati R. Mujumdar, C. J. Lewis, and A. S. Waggoner, in xe2x80x9cCyanine Dye Labelling Reagents: Sulfoindocyanine Succinimidyl Estersxe2x80x9d, Bioconjugate Chemistry, 4, 105, (1993) described the synthesis of hemicyanine intermediates for the preparation of sulfoindocyanine dyes active esters, useful as labelling reagents. One intermediate was obtained by condensing 1-ethyl-2,3,3-trimethyl[3H]indolium-5-sulfonate with N,Nxe2x80x2-diphenylformamidine in acetic acid for four hours. While the reported yield of the crude compound was 30%, the carboindocyanine dyes prepared from it were obtained only in 25% and 5% yields, after extensive purification by reverse phase HPLC chromatography. Similarly, the condensation of 1-ethyl-2,3,3-trimethyl[3H]indolium-5-sulfonate with malonaldehyde dianil hydrochloride in a mixture of acetic acid and acetic anhydride at reflux for four hours was said to produce the corresponding hemicyanine intermediate in an unreported yield. Again, the yields of the dicarboindocyanine dyes obtained from these intermediates were very low (5%). In fact, when Mank (Anal. Chem., 67, 1744) tried to synthesise the same dicarbocyanine label described in the previous reference he obtained a total yield of 18% of dicarbocyanines, from which the desired product was difficult to separate. He then devised an alternative approach based on 1,3,3-trimethoxypropene. Unfortunately, this chemical is no longer available commercially. Similar difficulties were encountered by us when trying to repeat the syntheses indicated above.
For these reasons it became necessary to investigate more carefully the technique employed to prepare the required hemicyanine intermediates and the properties of the latter. We thus discovered two main sources of trouble. The first was the formation of symmetrical indocyanine dye in variable and often erratic amounts in the condensation step. The other complication arose from the reversibility of this reaction. For example, when a pure sample of hemicyanine intermediate was exposed to the same conditions used for the formation of the asymmetric cyanine dye, namely to a base such as acetate, pyridine or triethylamine, formation of symmetric dye was observed. This phenomenon was more evident with the more reactive quaternised indolenines.
The steps we took to deal with these problems are described in details in the next section.
When a quaternised indolenine is reacted with N,Nxe2x80x2-diphenylformamidine in acetic acid, or acetic acid anhydride, or a mixture of these two solvents a hemicyanine is formed. This hemicyanine can be present in two forms, one in which the terminal anil group (NH-phenyl) is free and the other one where it is acetylated, NAc-phenyl, FIG. 3. The two forms have different UV-visible absorptions and can be easily differentiated. Even when acetic anhydride alone is used, some non acetylated hemicyanine is often formed. Similar observations apply to the reaction of quaternised indolenine with the vinilogs of N,N-diphenylformamidine, e.g. malonaldehyde dianils. Our initial aim in the optimisation of this reaction was the obtainment of only one of the two forms of hemicyanine, the acetylated form. We thought the addition of a stronger acetylating agent, such as acetyl chloride to the acetic anhydride solvent would be more effective in achieving the complete acetylation of the hemicyanine. Surprisingly, not only our assumption turned out correct, but, more importantly, the addition of acetyl chloride completely inhibited the formation of symmetric cyanine dye. Therefore, one aspect of our invention lies in the addition of variable amounts of acetyl chloride to the acetic anhydride solvent used for the preparation of hemicyanine. The amount of acetyl chloride can vary from 0.5% to 50%, with a preferred range from 1% to 20%.
In a further aspect of our invention, we developed a purification method of the intermediate hemicyanine by means of continuous extraction of the crude product with a suitable solvent. Such solvent should extract any unreacted N,N-diphenylformamidine or malonaldehyde dianil or substituted derivatives thereof, without contemporaneous dissolution of the hemicyanine. Preferred solvents are ethyl acetate, methylene chloride, chloroform, 1,1,1-trichloroethane and other chlorinated solvents. Methods for continues extraction include Soxhlet extraction or similar liquid-solid extractions, or liquid-liquid extractor; the latter can be in either of two forms, namely, the extracting liquid can be either lighter or heavier than the liquid containing the hemicyanine to be purified, either in solution or in a suspension. When the hemicyanine is present in a suspension, the density of the hemicyanine should be lower than the extracting solvent.
In yet another aspect of our invention, we designed a general strategy for the synthesis of asymmetric cyanine dyes to be used as labelling reagents. As we have seen previously, one of the complications present in these syntheses is the reversibility of the intermediate hemicyanines, especially when the second quaternised indolenine is less reactive then the first. In this case, large amount of symmetric hemicyanine, HET1-HET1 form, contaminating the desired HET1-HET2 product. The separation of the two products is often difficult to achieve, especially when the indolenine nuclei are similar. Of the different indolenines employed in these syntheses, the more reactive are those not bearing electron withdrawing groups in the benzene ring or with additional condensed benzene rings and with simple alkyl chains attached to the quaternary nitrogen. A decrease in reactivity is observed when these cyanines bear carboxylalkyl or sulfonatoalkyl chains or electron withdrawing groups in the benzene ring, such as sulfonic or carboxylic groups. A similar decrease in reactivity is also observed with cyanines bearing additional condensed benzene rings. The least reactive cyanines are those with additional condensed benzene rings bearing sulfonic groups and quaternised with sulfoalkyl or carboxylalkyl groups. In view of this behaviour we adopted the following strategy. First, we synthesised the hemicyanine with the less reactive indolenine according to the improved methods illustrated previously. These intermediate where then purified, when possible, by extraction of the impurities in an solid-liquid or liquid-liquid extractor. The purified hemicyanine intermediate was then reacted with the more reactive indolenine. Thus the reaction leading to the desired cyanine product was relative fast compared to the dissociation of the hemicyanine back to its starting materials. The products thus obtained were much more pure and their yields also increased significantly. Especially useful was found the combination of a hemicyanine made from a benzoindolenine bearing sulfonic groups and quaternised with a carboxylalkyl chain and an indolenine with sulfonic groups, but quaternised only with simple alkyl chains. This scheme is the opposite of those utilised according to known methods, especially those described in the above mentioned references, where the indocyanine intermediate is always synthesised from the more reactive indolenine, while the less reactive indolenine was added in the cyanine forming step.
In a further extension of this strategy, a more reactive malonaldehyde dianil reagents, 2-chloromalonaldehyde dianil hydrochloride was employed, especially when the indolenine to be reacted with it had very reduced reactivity.
The subject matter of the invention is defined by the appended claims.
In the claims, the term xe2x80x9csubstituted phenylxe2x80x9d is meant to include preferably phenyl with one or more substituents selected from alkyl and halogen atoms. Alkyl in the claims is preferably C1-C6 alkyl.
The following examples are simply meant to further illustrate specific applications of the present and are not intended to be construed as defining or limiting the scope of the invention.